Field of the Invention
This invention relates to photosynthetic microorganisms that convert inorganic carbon to fatty acids and secrete them into the culture medium, methods of production of fatty acids using such organisms, and uses thereof. The fatty acids may be used directly or may be further modified to alternate forms such as esters, reduced forms such as alcohols, or hydrocarbons, for applications in different industries, including fuels and chemicals.
Background Information
Photosynthetic microorganisms, including eukaryotic algae and cyanobacteria, contain various lipids, including polar lipids and neutral lipids. Polar lipids (e.g., phospholipids, glycolipids, sulfolipids) are typically present in structural membranes whereas neutral lipids (e.g., triacylglycerols, wax esters) accumulate in cytoplasmic oil bodies or oil globules. A substantial research effort has been devoted to the development of methods to produce lipid-based fuels and chemicals from photosynthetic microorganisms. Typically, eukaryotic microalgae are grown under nutrient-replete conditions until a certain cell density is achieved, after which the cells are subjected to growth under nutrient-deficient conditions, which often leads to the accumulation of neutral lipids. The cells are then harvested by various means (e.g., settling, which can be facilitated by the addition of flocculants, followed by centrifugation), dried, and then the lipids are extracted from the cells by the use of various non-polar solvents. Harvesting of the cells and extraction of the lipids are cost-intensive steps. It would be desirable to obtain lipids from photosynthetic microorganisms without the requirement for cell harvesting and extraction.
PCT publication numbers WO2007/136762 and WO2008/119082 describe the production of biofuel components using microorganisms. These documents disclose the production by these organisms of fatty acid derivatives which are, apparently, short and long chain alcohols, hydrocarbons, fatty alcohols and esters including waxes, fatty acid esters or fatty esters. To the extent that fatty acid production is described, it is proposed as an intermediate to these derivatives, and the fatty acids are therefore not secreted. Further, there is no disclosure of converting inorganic carbon directly to secreted fatty acids using a photosynthetic organism grown in a culture medium containing inorganic carbon as the primary carbon source. The present invention takes advantage of the efficiency of photosynthetic organisms in secreting fatty acids into the medium in order to recover these valuable compounds.
The invention includes the expression of heterologous acyl-ACP thioesterase (TE) genes in photosynthetic microbes. Many of these genes, along with their use to alter lipid metabolism in oilseeds, have been described previously. Genes encoding the proteins that catalyze various steps in the synthesis and further metabolism of fatty acids have also been extensively described.
The two functional classes of plant acyl-ACP thioesterases (unsaturated fatty acid-recognizing Fat A versus saturated fatty acid-recognizing FatB) can be clustered based on amino acid sequence alignments as well as function. FatAs show marked preference for 18:1-ACP with minor activity towards 18:0- and 16:0-ACPs, and FatBs hydrolyze primarily saturated acyl-ACPs with chain lengths that vary between 8-16 carbons. Several studies have focused on engineering plant thioesterases with perfected or altered substrate specificities as a strategy for tailoring specialty seed oils.
As shown in FIG. 1, fatty acid synthetase catalyzes a repeating cycle wherein malonyl-acyl carrier protein (ACP) is condensed with a substrate, initially acetyl-CoA, to form acetoacetyl-ACP, liberating CO2. The acetoacetyl-ACP is then reduced, dehydrated, and reduced further to butyryl-ACP which can then itself be condensed with malonyl-ACP, and the cycle repeated, adding a 2-carbon unit at each turn. The production of free fatty acids would therefore be enhanced by a thioesterase that would liberate the fatty acid itself from ACP, breaking the cycle. That is, the acyl-ACP is prevented from reentering the cycle. Production of the fatty acid would also be encouraged by enhancing the levels of fatty acid synthetase and inhibiting any enzymes which result in degradation or further metabolism of the fatty acid.
FIG. 2 presents a more detailed description of the sequential formation of acyl-ACPs of longer and longer chains. As shown, the thioesterase enzymes listed in FIG. 2 liberate the fatty acid from the ACP thioester.
Taking advantage of this principle, Dehesh, K., et al., The Plant Journal (1996) 9:167-172, describe “Production of high levels of octanoic (8:0) and decanoic (10:0) fatty acids in transgenic canola by overexpression of ChFatB2, a thioesterase cDNA from Cuphea hookeriana.” Dehesh, K., et al., Plant Physiology (1996) 110:203-210, and report “Two novel thioesterases are key determinants of the bimodal distribution of acyl chain length of Cuphea palustris seed oil.”
Voelker, T., et al., Science (1992) 257:72-74, describe “Fatty acid biosynthesis redirected to medium chains in transgenic oilseed plants.” Voelker, T., and Davies, M., Journal of Bacteriology (1994) 176:7320-7327, describe “Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase.”